Genome amplification and cellular senescence are hallmarks of human placenta development.

Genome amplification and cellular senescence are commonly associated with pathological processes. While physiological roles for polyploidization and senescence have been described in mouse development, controversy exists over their significance in humans. Here, we describe tetraploidization and senescence as phenomena of normal human placenta development. During pregnancy, placental extravillous trophoblasts (EVTs) invade the pregnant endometrium, termed decidua, to establish an adapted microenvironment required for the developing embryo. This process is critically dependent on continuous cell proliferation and differentiation, which is thought to follow the classical model of cell cycle arrest prior to terminal differentiation. Strikingly, flow cytometry and DNAseq revealed that EVT formation is accompanied with a genome-wide polyploidization, independent of mitotic cycles. DNA replication in these cells was analysed by a fluorescent cell-cycle indicator reporter system, cell cycle marker expression and EdU incorporation. Upon invasion into the decidua, EVTs widely lose their replicative potential and enter a senescent state characterized by high senescence-associated (SA) ß-galactosidase activity, induction of a SA secretory phenotype as well as typical metabolic alterations. Furthermore, we show that the shift from endocycle-dependent genome amplification to growth arrest is disturbed in androgenic complete hydatidiform moles (CHM), a hyperplastic pregnancy disorder associated with increased risk of developing choriocarinoma. Senescence is decreased in CHM-EVTs, accompanied by exacerbated endoreduplication and hyperploidy. We propose induction of cellular senescence as a ploidy-limiting mechanism during normal human placentation and unravel a link between excessive polyploidization and reduced senescence in CHM.

3D gait analysis, haemophilia joint health score, leg muscle laterality and biomarkers of joint damage: A cross-sectional comparative assessment of haemophilic arthropathy.

INTRODUCTION: 3D gait analysis has been proposed as a reproducible and valid method to assess abnormal gait patterns and to monitor disease progression in patients with haemophilia (PWH). AIM: This study aimed at comparing Gait Deviation Index (GDI) between adult PWH and healthy controls, and at assessing the agreement between outcome measures of haemophilic arthropathy. METHODS: Male PWH aged 18-49 years (prespecified subgroups: 18-25 vs 26-49 years) on prophylactic replacement therapy, and male healthy age-matched controls passed through a cross-sectional assessment panel. Besides the 3D gait analysis derived GDI, secondary outcomes included kinematic, kinetic and spatio-temporal gait parameters, the Haemophilia Joint Health Score (HJHS), electric impedance derived leg muscle laterality and inflammatory biomarkers. RESULTS: Patients with haemophilia (n = 18) walked slower, in shorter steps and accordingly with less functional range of motion in the hips and ankles, as compared to healthy controls (n = 24). Overall, PWH did not differ significantly in GDI and specific gait parameters. PWH had a higher mean HJHS (18.8 vs 2.6, P = .000) and leg muscle laterality (4.3% vs 1.5%, P = .004). A subgroup analysis revealed progressed gait pathology in PWH aged 26-49 years (not statistically significant). Leg muscle laterality was strongly correlated with HJHS (r = .76, P = .000), whereas GDI just moderately (r = -.39, P = .110). PWH had higher levels of the inflammatory markers CRP and IL-6. CONCLUSION: Progressed gait pathology was found in PWH, mainly those aged 26-49 years. Leg muscle laterality correlated strongly with HJHS and was identified as a promising tool for detecting progression and physiological consequences of haemophilic joint arthropathy.

Neuregulin-1-mediated ErbB2-ErbB3 signalling protects human trophoblasts against apoptosis to preserve differentiation.

During placentation, foetal trophoblasts invade deeply into maternal tissue to establish a foeto-maternal circulation. We have previously shown that extravillous trophoblast (EVT) lineage cells express ErbB2 and ErbB3, of which the potential as an oncogenic unit is well established. However, a physiological function of this receptor combination in humans remains a puzzling question. Here, we demonstrate neuregulin 1 (NRG1) expression and secretion by human decidual stromal cells. Stimulation of human primary trophoblasts with exogenous NRG1 induced phosphorylation of ErbB2, ErbB3 and related downstream effectors. Co-immunoprecipitation experiments confirmed the formation of ErbB2-ErbB3 dimers upon ligand engagement. Along this line, receptor knockdown and ErbB3 neutralization strongly diminished NRG1-dependent activation of the signalling complex. Functional studies revealed that NRG1 promotes EVT formation in placental explant cultures. Although, in the presence of NRG1, basal and camptothecin-induced trophoblast apoptosis was significantly repressed, this effect was abolished upon ErbB3 inhibition. Notably, camptothecin provoked a strong reduction of trophoblast cell column size, whereas NRG1-treated explants were refractory to the compound. Taken together, our findings newly identify a physiological function of the NRG1-ErbB2-ErbB3 axis in trophoblast survival during human placental development.

Response Profiling Using Shotgun Proteomics Enables Global Metallodrug Mechanisms of Action To Be Established.

Response profiling using shotgun proteomics for establishing global metallodrug mechanisms of action in two colon carcinoma cell lines, HCT116 and SW480, has been applied and evaluated with the clinically approved arsenic trioxide. Surprisingly, the complete established mechanism of action of arsenic trioxide was observed by protein regulations in SW480, but not HCT116 cells. Comparing the basal protein expression in the two cell lines revealed an 80 % convergence of protein identification, but with significant expression differences, which in turn seem to affect the extent of protein regulation. A clear-cut redox response was observed in SW480 cells upon treatment with arsenic, but hardly in HCT116 cells. Response profiling was then used to investigate four anti-cancer metallodrugs (KP46, KP772, KP1339 and KP1537). Proteome alterations were mapped to selected functional groups, including DNA repair, endocytosis, protection from oxidative stress, protection from endoplasmatic reticulum (ER) stress, cell adhesion and mitochondrial function. The present data suggest that knowledge of the mechanism of action of anti-cancer metallodrugs and improved patient stratification strategies are imperative for the design of clinical studies.

Trophoblast subtype-specific EGFR/ERBB4 expression correlates with cell cycle progression and hyperplasia in complete hydatidiform moles.

STUDY QUESTION: Do trophoblast subtypes differ in their expression of erythroblastic leukaemia viral oncogene homologue (ERBB) receptor family members and responsiveness towards specific growth factor ligands? SUMMARY ANSWER: Our data reveal a reciprocal expression pattern of epidermal growth factor receptor (EGFR)/ERBB4 in proliferative and ERBB2/ERBB3 in invasive trophoblast subtypes, as well as a restricted responsiveness to epidermal growth factor (EGF) and heparin-binding (HB)-EGF. WHAT IS KNOWN ALREADY: EGFR is expressed by villous cytotrophoblasts (vCTBs), but absent from extravillous trophoblasts (EVTs), which specifically up-regulate ERBB2. STUDY DESIGN, SIZE, DURATION: Tissue samples of human first trimester placentae (n = 50) and deciduae (n = 5) obtained from elective pregnancy terminations were used to study trophoblast subtype-specific ERBB receptor expression and responsiveness to recombinant human EGF and HB-EGF. Age-matched complete hydatidiform mole (CHM) placentae (n = 12) were assessed for EGFR and ERBB4 expression in proliferation-competent regions. PARTICIPANTS/MATERIALS, SETTING, METHODS: ERBB receptor expression was analysed in primary trophoblast cell isolates by means of microarray, quantitative real-time PCR and western blotting, as well as immunofluorescence stainings of placental and decidual tissue sections. EGF and HB-EGF were tested for their potential to activate ERBB receptors in purified EGFR(+) and HLA-G(+) trophoblasts. 5-Ethynyl-2'-deoxyuridine incorporation assays were performed to study the effect of both ligands on the proliferative capacity of primary trophoblasts as well as of vCTBs and proximal cell column trophoblasts (pCCTs) in placental floating explants. Finally, the average number of EGFR(+) vCTB and pCCT layers was determined in CHM placentae and compared with healthy age-matched controls. MAIN RESULTS AND THE ROLE OF CHANCE: Proliferative vCTBs and pCCTs co-express EGFR and ERBB4, but are devoid of ERBB2 and ERBB3. In contrast, HLA-G(+) trophoblast subtypes exhibit an EGFR/ERBB4(-) and ERBB2/ERBB3(+) phenotype. EGF and HB-EGF activate EGFR, ERBB4, AKT and extracellular signal-regulated kinase 1/2 in EGFR(+) primary trophoblasts; however, they do not show an effect on HLA-G(+) EVTs. Both ligands strongly induce cell cycle progression in primary trophoblasts (P < 0.05) and placental explant-associated vCTBs (P < 0.05) and pCCTs (P < 0.05). Notably, EGFR(+) vCTB (P < 0.0001) and pCCT (P < 0.0001) layers are significantly expanded in CHM placentae when compared with healthy controls. LIMITATIONS, REASONS FOR CAUTION: Cells were removed from their physiological context and may therefore respond differently to various stimuli. WIDER IMPLICATIONS OF THE FINDINGS: In this study we define EGFR as a marker for proliferative trophoblast subtypes within the human placenta. Manipulation of EGFR signalling might thus offer a promising therapeutic avenue for the treatment of molar pregnancies associated with trophoblast hyperplasia. STUDY FUNDING/COMPETING INTERESTS: This study was supported by the Austrian Science Fund (grant P-25187-B13 to J.P.). There are no competing interests to declare.